Abstract

The diagnosis of autoimmune hemolytic anemia (AIHA) can be made

with a stepwise approach that aims to identify laboratory and clinical
evidence of hemolysis and then determine the immune nature of
hemolysis with the direct anti-globulin test. Once alternative causes for
these findings have been excluded, AIHA is established, and the clinician
must search for secondary causes, as well as identify the type of AIHA.
Rituximab is now the preferred second-line treatment for primary warm
AIHA and first-line treatment for primary cold agglutinin disease (CAD),
either as monotherapy or combined with bendamustine. Complement
inhibitors have shown utility in stabilizing AIHA patients with acute
severe hemolysis. Future prospects are discussed and include the C1s
inhibitor BIVV009 (sutimlimab) that is now entering phase 3 studies for
CAD.

Learning Objectives
 Understand how AIHA and its subtypes can be diagnosed through a
combination of clinical assessment and laboratory tests
 Describe the latest advances in treatment of primary warm AIHA
and cold agglutinin disease
Go to:

Approach to the diagnosis of autoimmune hemolytic anemia

Autoimmune hemolytic anemia (AIHA) is a decompensated acquired

hemolysis caused by the host’s immune system acting against its own
red cell antigens. Consequent complement activation can impact the
clinical picture and is an emerging target for therapeutic approaches.

When a patient presents with anemia, a stepwise approach should be

followed. Initial simple investigations (Table 1) will first alert the
physician to the suggestion of hemolysis as the cause of the anemia.
These include a normo-/macrocytic anemia, raised reticulocyte count,
raised unconjugated bilirubin, reduced haptoglobin, and blood smear
with polychromasia or more specific features, such as spherocytes or
agglutination (Figure 1). Although the typical pattern is presented, none
of these tests are fully sensitive or specific for hemolysis. For example,
liver disease can increase lactate dehydrogenase (LDH) and reduce
haptoglobin. The bilirubin may be normal with milder hemolysis, and
spherocytes are not always visible. Reticulocytopenia can occur in AIHA,
secondary to bone marrow infiltration by a lymphoproliferative disorder
or to parvovirus B19 infection. However, reticulocytopenia is also
observed in a significant minority of patients at presentation (20% in 1
series1), despite erythroid hyperplasia in the marrow. This may be
caused by immune attack on late-stage erythroid precursors or reflect a
lag in marrow responsiveness, but it can sometimes persist and predict a
more severe clinical course.2 A significantly raised LDH, red cell
fragments on smear, or the presence of urinary hemosiderin suggests a
predominant intravascular component to the hemolytic process. There
are fewer causes of intravascular hemolysis; therefore, these can be very
useful for subsequent investigations (Table 2). Once hemolysis is
confirmed, further investigation is needed to establish whether that
hemolysis is immune, principally by the direct anti-globulin test (DAT).
The standard DAT demonstrates that immunoglobulin G (IgG) and/or
complement (usually C3d) is bound to the red cell membrane.
Autoantibodies can also be of IgM and IgA classes, and, in some
circumstances, an extended DAT panel can be used to detect these.

Table 1.
Investigations in patients presenting with AIHA
Diagnostic tests Indication for tests

Primary evaluation

 Hemolytic screen

  FBC, blood smear, LDH, haptoglobin, bilirubin,

DAT, reticulocyte count with or without urine for
hemosiderin or urine dipstick for microscopy
Diagnostic tests Indication for tests

 Detection of underlying disorders (investigation of

AIHA)

  Serum Igs and electrophoresis with

immunofixation*

  HIV, HBV, HCV

  Anti-dsDNA, ANA

  CT chest, abdomen, and pelvis

Additional investigation in selected patients with

AIHA

CAD, age ≥ 60 years, features in history,

 Bone marrow examination examination, FBC or smear suggesting
possible marrow infiltration

If pregnant or thrombocytopenic, to exclude

 U&E, LFT, clotting, BP, urine dipstick
DIC or pregnancy-associated TMA

Dependent on symptoms, travel history, and

 Infection screening
age

 Peripheral T-cell subsets, creatinine, LFT, clotting All children and if suspected Evans syndrome

 Parvovirus, hematinics If reticulocytopenia

Additional serological investigation in selected

patients with AIHA

 DAggT If DAT positive for C3d ± IgG

 Cold antibody titer If DAggT positive

 Monospecific DAT for IgM, G, A, C3d If DAT-negative AIHA suspected

If (monospecific) DAT-negative AIHA

 Red cell eluate
suspected

 Donath Landsteiner If DAT is positive for C3d ± IgG and

i) DAggT-negative or insignificant CAs and

 Diagnostic tests Indication for tests

ii) Age < 18 y or hemoglobinuria or cold-

associated symptoms or atypical serology

If clinical significance of cold autoagglutinin

 Thermal range of cold antibody
unclear

Open in a separate window

ANA, antinuclear antibody; BP, blood pressure; CAD, cold agglutinin disease; CAs, cold
agglutinins; CT, computerized tomography; DAggT, direct agglutination test; DIC,
disseminated intravascular coagulation; dsDNA, double-stranded DNA; FBC, full blood
count; HBV, hepatitis B virus; HCV, hepatitis C virus; LFT, liver function tests; TMA,
thrombotic microangiopathy; U&E, urea and electrolytes.
*
If a cold autoantibody suspected, keep sample at 37°C until serum has been separated.

Modified from Hill et al, with permission.12

Figure 1.

Blood film appearances in patients with AIHA (both using May Grunwald Giemsa stain). (A)
Spherocyte in a patient with warm AIHA (original magnification, ×100). (B) Red cell
agglutination in a patient with cold agglutinin disease (×40).
Table 2.
Causes of intravascular hemolysis
Complement fixation (eg)

 Paroxysmal nocturnal hemoglobinuria

 Paroxysmal cold hemoglobinuria

 Acute hemolytic transfusion reactions due to mismatched blood

 Some autoimmune hemolytic anemias if complement fixation allows terminal complement

activation (membrane attack complex formation) on the surface of red blood cells causing lysis

Mechanical trauma (eg)

 Cardiac valves

 Microthrombi in circulation - DIC, MAHA (TTP, HUS, aHUS)

 March hemoglobinuria

Toxic or exogenous factors (eg)

 Infections: Babesia, Clostridium, Leptospira, Falciparum malaria

 Spider bites

 Snake venoms

 Copper or zinc toxicity

 Hypotonic solutions

Open in a separate window

aHUS, atypical hemolytic uremic syndrome; DIC, disseminated intravascular coagulation;

HUS, hemolytic uremic syndrome; MAHA, microangiopathic hemolytic anemia; TTP,
thrombotic thrombocytopenic purpura.

After identifying hemolytic anemia with a positive DAT, hopefully

obvious causes, such as a delayed transfusion reaction from a recent
transfusion, alloimmune hemolysis following solid organ or allogeneic
stem cell transplantation, drug-induced immune hemolysis, or hemolytic
disease of the newborn, will be quickly identified by clinical assessment.
When considering alternatives, it should be remembered that a positive
DAT can occur as a result of other processes, such as passive deposition
of antibodies or immune complexes in liver disease, chronic infection,
malignancy, systemic lupus erythematosus (SLE), renal disorders, and
following drug therapies, such as IV Ig or anti-thymocyte globulin. If no
alternative cause is identified, a diagnosis of AIHA can be made. AIHA can
occur infrequently with a negative DAT by standard techniques (eg, due
to a low-affinity antibody or an IgA-only antibody). A diagnosis of DAT-
negative AIHA can be made following careful exclusion of alternative
causes of hemolysis, and confirmation by a sensitive technique where
available, and is supported by a response to steroid therapy.

The next step is to investigate for an underlying associated condition,

which is found in ∼50% of patients, along with further serological tests
to determine the type of AIHA, because the approach to treatment differs
(Table 1). A suggested diagnostic pathway for AIHA is shown in Figure 2,
although it is recognized that there are exceptions (eg, a low-titer cold
antibody can sometimes be clinically significant). Therefore, a final
diagnosis may require synthesis of the clinical picture and advice from a
specialist reference laboratory.
Figure 2.

Diagnostic pathway for AIHA. DAggT, direct agglutination test; DIIHA, drug-induced
immune hemolytic anemia; HA, hemolytic anemia; HDN, hemolytic disease of the newborn;
HTR, hemolytic transfusion reaction; PLS, passenger lymphocyte syndrome; RT, room
temperature. *The final diagnosis of CHAD or mixed AIHA is based on the overall clinical
†
picture, including supportive serological findings.  For example, the thermal amplitude.
**Saline-suspended red cells and patient’s serum at room temperature for 30 to 60
12
minutes. Adapted from Hill et al with permission.
Approximately 65% of patients have a warm AIHA; this can be diagnosed
in patients with a consistent clinical picture and a DAT positive for IgG
only or when DAT is positive for C3d ± IgG, when a clinically significant
cold antibody has been excluded.

Primary cold agglutinin disease (CAD) is caused by an underlying

lymphoproliferative bone marrow disorder, and its pathophysiology is
described elsewhere.3 Hemolysis is caused by IgM antibodies that are
most active in vitro at low temperatures. In vivo, these antibodies bind
red cells in the cooler peripheral circulation, causing agglutination that
leads to acrocyanosis or Raynaud’s disease in some patients. IgM binds
C1q on the red cell membrane, activating the classical complement
pathway but disassociating from the red cells centrally, leading to a DAT
that is characteristically positive for only C3d, although a weak positive
IgG is sometimes also detected. This process leads to extravascular
removal of C3b-coated red cells by the reticuloendothelial system;
however, exacerbations can activate C5, leading to intravascular
hemolysis. In patients with a DAT positive for C3d ± IgG, a direct
agglutination test can be performed as a screening test in the local
transfusion laboratory, and a clinically significant cold agglutinin can be
excluded if saline-suspended normal red cells are not agglutinated by the
patient’s serum after incubation at room temperature for 30 to 60
minutes. Patients with positive screening can then have antibody titers
assessed. CAD can be diagnosed in patients with AIHA and a DAT
positive for C3d ± IgG, with a consistent clinical picture and a high-titer
cold reactive antibody (titer ≥ 1:64 at 4°C). Assessment of the antibodies’
thermal amplitude can be useful and is usually ≥30°C when red cells are
suspended in 30% bovine albumin. Serum electrophoresis will usually
detect a monoclonal paraprotein, typically IgMκ, but serum must be kept
at 37 to 38°C from the time of sampling until serum is removed from the
clot.

Cold agglutinin syndrome (CAS) can be diagnosed in patients with

laboratory criteria consistent with a clinically significant cold antibody
that occurs in association with secondary disorders, such as infection,
SLE, or aggressive lymphoma.
Mixed AIHA is caused by a combination of a warm IgG antibody and a
cold IgM antibody. The DAT is usually positive for IgG and C3d. Cold-
associated symptoms rarely appear, and the cold antibody may have a
low antibody titer (eg, <1:64) but with a thermal amplitude up to 30 to
37°C. It can be diagnosed with a DAT positive for IgG and C3d, a cold
antibody with a thermal amplitude ≥ 30°C, and an appropriate clinical
picture.

Paroxysmal cold hemoglobinuria (PCH) usually occurs in children. The

hemolysis can be severe and intravascular but is typically transient
following infection. PCH is caused by a biphasic IgG antibody that binds
to red cells at low temperature and causes complement-mediated lysis as
the temperature is increased. It can be diagnosed in patients with AIHA
and a positive Donath-Landsteiner test. Testing should be considered in
patients with AIHA and a DAT positive for C3d ± IgG (the DAT is
sometimes negative), when CAD has been excluded, and there is
hemoglobinuria, cold-associated symptoms, atypical serological features,
or patients younger than 18 years old.

Autoantibody specificity in PCH is usually anti-P, in contrast to CAD, in

which specificity is usually anti-I, but sometimes it is anti-i or anti-PR.
Warm AIHA autoantibodies are usually directed against a high incidence
antigen on the red cell surface, but ∼3% have specificity (eg, anti-e).
Criteria for the diagnosis of AIHA and its subtypes, as well as definitions
of treatment response, have varied among studies. An international
consensus on terminology and response criteria would be useful to
increase comparability between clinical trials.

Go to:

Treatment of AIHA

Secondary AIHA
British guidelines on secondary AIHA have recently been published.4 In
adults, the most common associations are hematological malignancy,
infection, and autoimmune disorders. A broad strategy is to treat the
underlying condition according to best practice. Successful treatment
may also improve the AIHA, but that is not always the case because the
strength of the association varies. If the associated condition does not
require treatment, AIHA can usually be approached in a similar fashion
to primary AIHA, although treatment decisions must be individualized.
For example, when AIHA is associated with some disorders, such as SLE,
the burdens and benefits of splenectomy are less favorable compared
with primary warm AIHA. Evan’s syndrome, in which AIHA is associated
with immune thrombocytopenia (ITP), has important differences from
primary AIHA, with regard to the exclusion of alternative causes of a
bilineage cytopenia, identifying secondary causes such as autoimmune
lymphoproliferative syndrome, and the management of its often
relapsing/remitting clinical course.

B-cell malignancies are a common association (eg, AIHA occurs in ∼5-

10% of patients with chronic lymphocytic leukemia [CLL]). In patients
with active CLL that also requires treatment, combinations, such as
rituximab, cyclophosphamide, and dexamethasone (RCD); rituximab,
cyclophosphamide, vincristine, and prednisolone (R-CVP); and
bendamustine and rituximab (BR), appear to be effective. New targeted
therapies, such as obinutuzumab, ibrutinib, idelalisib, and venetoclax,
could potentially be beneficial for patients with AIHA; however, vigilance
is needed because of potential for development of drug-induced immune
cytopenias, as previously observed with single agent fludarabine.5 This
issue has also been raised with ibrutinib, but emergent AIHA during
ibrutinib therapy appears to be an expression of CLL activity rather than
drug-induced hemolysis, and treatment can be administered in patients
with a history of AIHA.5,6

General strategies for primary AIHA

All patients

 Treat any identified underlying cause, such as infection.

 Erythropoiesis increases in response to hemolysis, resulting in
greater demand for folate. Folic acid is a well-tolerated supplement
to prevent a deficiency that could exacerbate the anemia.
 Compatibility testing for patients requiring transfusion focuses on
identifying the patient’s ABO CcDEe and K status, as well as the
presence of alloantibodies. This can be achieved by special
techniques, such as auto- or alloadsorbtion (warm autoantibodies)
or prewarming screening cells and patient’s plasma separately to
37°C before use in the indirect anti-globulin test (cold
autoantibodies).7 Genotyping can be useful, particularly for
complex cases in which it is desirable to match for an extended
phenotype. Clinicians should be aware that full compatibility
testing can take 4 to 6 hours and that the units provided will not be
labeled as “compatible,” because autoantibodies from unmodified
patient plasma are likely to bind donor red cells in an indirect anti-
globulin test cross-match. Without a history of transfusion or
pregnancy, the patient is unlikely to have an alloantibody. If anemia
is life threatening, transfusion of ABO Rh and K matched red cells
may be preferable to waiting for full-compatibility testing.
 Venous thromboembolism prophylaxis: thrombosis risk with
hemolysis is still underestimated but remains an important cause
of morbidity and mortality in AIHA. In some series, venous
thromboembolism events occurred in up to 20% to 25% of
patients, usually when hemolysis was active. Larger studies have
shown higher rates of venous and arterial thrombosis in patients
with AIHA8,9 and, more recently, in those with CAD
specifically.10 Perhaps not surprisingly, the risk increases with
higher LDH levels (the role of intravascular hemolysis and
thrombosis risk has been discussed previously11), as well as in
patients with previous splenectomy. Thromboprophylaxis with
low-molecular-weight heparin is recommended for in-patients
with an acute exacerbation of hemolysis and should be considered
in ambulatory patients during severe exacerbations (hemoglobin <
85 g/L).12
CAD patients

 In CAD patients, intravascular hemolysis can be triggered by

bacterial or other febrile illnesses and should be treated promptly.
 Patients with CAD should avoid exposure to cold weather where
possible and dress to protect the distal extremities.
 While in hospital, the patient should be kept warm, especially in
the perioperative period. Cardiopulmonary bypass presents a
particular challenge related to the usual practice of systemic
cooling and cold cardioplegia. However, systemic normothermia
and warm cardioplegia is a recognized method, and adjunctive
eculizumab has been successfully used to cover bypass surgery.13
 Patients should not receive cold fluids IV, and use of an in-line
blood warmer is recommended.
Patients receiving steroids

 Because of an increased risk for upper gastrointestinal

complications, antacid therapy, such as a proton pump inhibitor, is
recommended in patients taking steroids, if there is an additional
risk factor, such as previous peptic ulcer disease, age ≥ 60 years,
concomitant nonsteroidal anti-inflammatory
drug/anticoagulant/aspirin use, or concomitant thrombocytopenia.
 Fracture risk is increased in patients receiving glucocorticoids for
≥3 months (prednisolone ≥ 7.5 mg/d). It is recommended that
these patients are advised about lifestyle modification (smoking
cessation, limiting alcohol intake to ≤2 U/d, and engaging in regular
weight-bearing exercise) and maintain adequate daily vitamin D
(600-800 IU) and calcium (1000-1200 mg) intake through diet, if
possible, or supplements, if needed.14 In addition, for adults ≥40
years of age, the initial absolute fracture risk should be estimated
using the online tool FRAX
(https://www.shef.ac.uk/FRAX/tool.jsp), as well as bone mineral
density testing, if available, to determine whether an oral
bisphosphonate should also be prescribed.14 However, some
guidelines also recommend that men older than 50 years of age and
 postmenopausal women are at sufficient risk that treatment may
be considered without further assessment.15

Primary warm AIHA

Although most patients require treatment, watchful waiting may be

appropriate in a minority of patients with mild asymptomatic anemia.
For many years, the standard first-line therapy has been glucocorticoids,
typically prednis(ol)one at a starting dose of 1 mg/kg. The response rate
is ∼80%, but ∼60% of these lose their response upon weaning or
discontinuing steroids.16,17 Although AIHA is rare, with a weak evidence
base to guide treatment, 2 phase 3 prospective randomized trials have
compared predniso(lo)ne with predniso(lo)ne-rituximab as first-line
treatment for warm AIHA, although not all cases were primary. A Danish
study randomized 64 patients to prednisolone or prednisolone with
rituximab (375 mg/m2 weekly for 4 weeks). The response rate to
prednisolone-rituximab was significantly higher (75% vs 36%; P = .003)
at 12 months, with no difference in adverse reactions.18 A French study
randomized 32 patients to prednisone-placebo vs prednisone-rituximab
(1 g on days 1 and 15). Again, the primary end point of overall response
at 12 months was significantly higher (75% vs 31%; P = .032) in patients
receiving rituximab, without excess adverse events.19 Ideally, larger
studies of longer duration, powered to determine the impact of
rituximab on transfusion requirement, cumulative steroid exposure, and
health-related quality of life, would help to inform the first-line standard
of care. However, given that steroids are often used to rescue relapsing
patients, and prolonged steroid use carries a significant risk for infection,
diabetes, and fracture,17,20 the use of first-line rituximab with a relatively
short course of steroids has been suggested for elderly patients or those
with comorbidities.19

In patients receiving first-line steroids, the long-standing second-line

treatment of choice was splenectomy.21 However, recent United
Kingdom12 and French22 guidelines have instead favored rituximab. This
reflects the relatively large number of published case series that have
increased certainty in its effectiveness and tolerability. In a meta-
analysis of 154 patients, the overall response rate of warm AIHA to
rituximab was 79%, although not all cases were primary, and some
received concomitant steroids.23 Adverse events occurred in 38 of 364
(14%) AIHA patients receiving rituximab, including 18 severe infections.
Approximately 25% to 50% of responding patients will relapse within 2
to 3, whereas long-term response rates are unknown.12

Approximately 70% of patients with primary warm AIHA respond to

splenectomy,12 and this continues to be a useful second-line option when
economic restraints limit access to rituximab.24 The infective and
thrombotic complications following splenectomy are well recognized.
Small series suggest ∼30% relapse in the short term,17,25 but unlike
splenectomy for ITP, the long-term durability of remission is unknown.
Alternative, relatively nontoxic immunosuppressive therapies, such as
mycophenolate mofetil, azathioprine, and cyclosporin, can also be used
as third-line treatment. Response rates are difficult to estimate because
evidence of efficacy is limited to small series that often lack detail, but
they are likely to be lower than with rituximab or splenectomy.

Patients who are steroid sensitive, but refractory or relapsing after third-
line therapies, may tolerate longer-term treatment with low-dose
predniso(lo)ne (≤10 mg), which can be used in conjunction with a
steroid-sparing agent.26 Alternatives with greater potential for toxicity
include cyclophosphamide,27,28 alemtuzumab,29 and hematopoietic stem
cell transplantation.30

Rescue therapies 

Patients can take time to respond to immunosuppressive therapy (eg,

median of 3-6 weeks for rituximab for warm AIHA).12 Therefore, those
with severe transfusion-dependent hemolysis may need rescue therapy.
This may be with steroids if the patient is known to be sensitive.
Alternatively, approximately one third of patients will respond to IV Ig,
with better responses observed in the most severe cases (hemoglobin ≤
70 g/L). The clinical severity of AIHA is influenced by the marrow’s
compensatory response, and reticulocytopenia has been observed,
particularly in children and severe cases, possibly as a result of
autoantibodies acting against erythroid precursors.31 This observation
provides rationale for the empirical use of recombinant erythropoietin
when hemolysis is severe with a poor reticulocyte response; however,
parvovirus, hematinic deficiency, and marrow infiltration should also be
considered. Emergency splenectomy or partial splenic embolization in
unfit patients has been used acutely to treat severe hemolysis.

Autoreactive IgG and IgM can be produced in warm AIHA; when bound
to the erythrocyte, both are able to bind C1q and activate the classical
complement pathway. C1 esterase inhibitor (C1-INH) is a commercially
available inhibitor of the classical pathway licensed for hereditary
angioedema with a favorable safety profile. C1-INH has been used to
improve response to transfusion in a patient with severe IgM warm
AIHA.32 Furthermore, 4 patients with CAD or mixed AIHA and a C3d-
positive DAT all responded to the C1-INH BERINERT (20 μg/kg daily for
6-20 days given with prednisolone, with or without
rituximab).33 Although data are sparse, C1-INH may have a role in
stabilizing severe hemolysis if the DAT is positive for C3d.

Future prospects 

Future prospects for primary warm AIHA include inhibition of the

following:

 B cells with alternative anti-CD20 monoclonal antibodies (mAbs)

(eg, obinutuzumab)
 Mammalian target of rapamycin (eg, sirolimus)
 Proteasomes (eg, bortezomib)
 Spleen tyrosine kinase (Syk) (eg, fostamatinib)
 Neonatal Fc receptor (FcRn)
 Bruton tyrosine kinase (BTK)
 Complement pathways

Following the success of rituximab, additional anti-CD20 mAbs were

developed, primarily to target B-cell malignancies. These vary in terms of
their antibody effector functions, such as complement-dependent
cytotoxicity, directly induced nonapoptotic cell death, and FcγR-
dependent mechanisms. Third-generation mAbs have been engineered
to enhance effector functions (eg, removal of the Fc glycan fucose to
increase FcγRIIIA binding affinity; obinutuzumab).34 Other anti-CD20
mAbs are being explored in autoimmune diseases, such as ITP
(veltuzumab) and multiple sclerosis (ublituximab), but their role in AIHA
awaits investigation. Another novel strategy aiming to reduce relapse
would be maintenance treatment with an anti-CD20 mAb.

The mammalian target of rapamycin inhibitor sirolimus, which increases

T-regulatory cells and induces apoptosis in abnormal lymphocytes, has
been used successfully in children to treat autoimmune
lymphoproliferative syndrome, posttransplant AIHA, and 4 of 4 patients
with refractory primary AIHA.35

The proteasome inhibitor bortezomib has been used to treat antibody-

mediated hematological disorders, including thrombotic
thrombocytopenic purpura, acquired hemophilia, and warm AIHA. It
causes apoptosis of antibody-producing plasma cells, downregulates NF-
κB–mediated proinflammatory signaling, depletes autoreactive T-helper
cells, and interferes with antigen presentation.36 Long-lived autoreactive
plasma cells have been shown to persist in the spleen of warm AIHA
patients who failed to respond to rituximab.37 In a retrospective study, 3
of 4 refractory patients with warm AIHA achieved at least a partial
response to bortezomib, although all patients were receiving
concomitant therapies, and prospective studies are needed.36

Binding of FcγR on inflammatory cells causes activation of Syk, which

leads to phagocytosis and immune activation. The Syk inhibitor
fostamatinib achieved a stable response in 18% of ITP patients treated in
2 phase 3 randomized controlled trials,38 and in the interim report of a
phase 2 trial in warm AIHA (ClinicalTrials.gov identifier NCT02612558),
9 of 17 (53%) patients responded to fostamatinib (150 mg, twice daily).39
FcRn is a widely expressed receptor that is able to bind and transport
IgG antibodies within and between cells. Endosomal FcRn prolongs the
half-life of IgG by recycling antibody that would otherwise undergo
lysosomal degradation. FcRn inhibitors are being studied in a variety of
autoimmune conditions with the aim of increasing catabolism of
pathogenic IgG. A phase 1 study of the FcRn inhibitor SYNT001 in warm
AIHA is underway (NCT03075878).

BTK is a signaling element downstream of the B-cell and Fcγ receptors.

The oral BTK inhibitor PRN1008 is being studied in ITP (NCT03395210),
but it might also be applicable to AIHA.

APL-2, an inhibitor of C3, is being evaluated in an open-label phase 2

study of patients with warm AIHA and CAD (NCT03226678).

Primary CAD

Patients with mild asymptomatic anemia can be monitored without

specific treatment, which should be reserved for symptomatic anemia,
severe circulatory symptoms, or transfusion dependence. Because
exacerbations can be transient, a period of support with transfusion may
be useful before determining the need for pharmacological intervention.

First-line treatment is usually rituximab (375 mg/m2 weekly for 4

weeks). Approximately 50% responded in 2 prospective case series, but
complete responses were rare.3 This lower response rate compared with
warm AIHA may be due to the presence of an underlying clonal
lymphoproliferative disorder and/or because the response criteria used
required improvement in cold associated symptoms and the clonal
disease, as well as hemolysis. Unfortunately, the median response
duration was only 11 months (Table 3), although 6 of 10 responding
patients also responded to a second course.40

Table 3.
Comparison of Nordic studies of rituximab and rituximab combination therapies for CAD
 Fludarabine-
BR (n = 45)45 Rituximab (n = 27)40
rituximab (n = 29)46

Rituximab: 375
Rituximab: 375 mg/m2 day 1
mg/m2 day 1
Bendamustine: 90 Rituximab: 375
Fludarabine (oral): 40
Regimen mg/m2 days 1 and 2, 4 mg/m2 day 1 (weekly for
mg/m2
cycles at 4 wk)
days 1 to 5, 4 cycles
a 28-d interval
at a 28-d interval

Response rate, %

 Overall response 71 76 52

 Complete
40 21 4
response

Median time to
response (range), 1.9 (0.25-12) 4.0 (0.3-6.0) 1.5 (0.5-4.0)*
mo

Sustained response

77% at median FU 33
 % 91% at median FU 36 mo Not reported
mo

 Median response Observed = 11 mo

Observed = 32 mo Estimated > 66 mo
duration (range 2-42)*

41% G3 or G4 3% G4 hematologic
Tolerance 33% G3 or G4 neutropenia
hematologic toxicity toxicity

59% G1 to G3
11% infections 3% G1 infection*
infections

1 fatal pneumonia at
1 fatal pneumonia at 5 mo
9 mo

1 fatal episode of
hypothermia during
rituximab

Open in a separate window

Response criteria for all 3 studies were the same.

FU, follow-up; G, grade.
*
Values are calculated from 37 treatment episodes in 27 patients.

Rituximab has been combined with bendamustine (BR) or fludarabine in

2 prospective uncontrolled studies (Table 3). Although the studies are
not directly comparable, the response criteria used were the same, and
there appeared to be a higher rate of complete remission and fewer
infections in patients receiving BR. Combination therapy provides more
opportunity for a durable response but with a greater risk for toxicity.
BR is a suitable second-line option for fit patients or a first-line option in
selected patients with severe disease.

A prospective open-label phase 2 study (NCT01696474) reported on 19

previously treated CAD patients who received a single course of
bortezomib (1.3 mg/m2) on days 1, 4, 8, and 11.41 Improved hemolysis
was documented in 6 of 19 patients, including complete resolution in 3.
Response was sustained in 4 of 6 patients at a median of 16 months
(range, 10-31). Some activity against the underlying B-cell clone was
observed, with a <50% decrease in monoclonal Ig from pretreatment
levels in 3 of 13 evaluable patients and a reduction in marrow B-cell
lymphoid infiltrate >50% in 2 of 5 cases. Although further studies are
needed, therapeutic options for CAD are limited, and bortezomib may be
a suitable second-line option in patients who are not fit for combination
therapy, such as BR or rituximab plus fludarabine.3

Emergency treatment 

CAD patients usually take several months to respond to rituximab-based

therapy (Table 3), whereas hemolysis can be severe and intravascular. In
addition to blood transfusion, daily or alternative-day plasma exchange
of 1 to 1.5 times the plasma volume with albumin has been used as a
bridging therapy, although evidence is mixed and largely confined to
case reports. Although not recommended for routine treatment of CAD,
steroids may have a limited role as rescue therapy in severe cases.
Patients with acute severe intravascular hemolysis may also respond to
the C5 inhibitor eculizumab, which blocks the terminal complement
pathway. Its activity in CAD is supported by case reports and a
prospective study of 13 patients who received 6 months of treatment,
with a reduction in transfusion requirements and LDH. 42

Future prospects 

Given the mechanism of hemolysis outlined previously, inhibition of the

classical complement pathway is a rational and promising therapeutic
approach for CAD. Treatment would need to be maintained to have a
continuing effect and is not expected to improve cold-associated
symptoms, which result from agglutination of antibody-bound red cells
upstream of complement activation. In a phase 1b study, 6 CAD patients
received a 10 mg/kg loading dose of the anti-C1s antibody BIVV009
(sutimlimab) and then received 60 mg/kg 1 to 4 days later, followed by 3
weekly doses of 60 mg/kg. Treatment was well tolerated, and all 6
patients responded with a mean increase in hemoglobin of 43 g/L. On-
going responses were observed in a named patient program using a fixed
biweekly dose of 5.5 g with up to 18 months of observation.43 Phase 3
CAD clinical trials (NCT03347396, NCT03347422) are now open.

The C3 inhibitor APL2 is being evaluated in warm AIHA and CAD, as

previously discussed.

Many more complement inhibitors are under development for various

clinical indications and have been recently reviewed.44

Given the clonal nature of CAD, newer targeted therapies for B-cell
malignancies, such as the BTK inhibitor ibrutinib and B-cell receptor
pathway–modulating drugs (eg, idelalisib and venetoclax) might have
activity in CAD, although the hypothesis awaits investigation. 3

Go to:

References
1. Liesveld JL, Rowe JM, Lichtman MA. Variability of the erythropoietic response in
autoimmune hemolytic anemia: analysis of 109 cases. Blood. 1987;69(3):820-826.
[PubMed] [Google Scholar]
2. Fattizzo B, Zaninoni A, Nesa F, et al.. Lessons from very severe, refractory, and
fatal primary autoimmune hemolytic anemias. Am J Hematol. 2015;90(8):E149-
E151. [PubMed] [Google Scholar]
3. Berentsen S. How I manage patients with cold agglutinin disease. Br J Haematol.
2018;181(3):320-330. [PubMed] [Google Scholar]
4. Hill QA, Stamps R, Massey E, Grainger JD, Provan D, Hill A; British Society for
Haematology Guidelines. Guidelines on the management of drug-induced immune
and secondary autoimmune, haemolytic anaemia. Br J Haematol. 2017;177(2):208-
220. [PubMed] [Google Scholar]
5. Molica S, Polliack A. Autoimmune hemolytic anemia (AIHA) associated with
chronic lymphocytic leukemia in the current era of targeted therapy. Leuk Res.
2016;50:31-36. [PubMed] [Google Scholar]
6. Montillo M, O’Brien S, Tedeschi A, et al.. Ibrutinib in previously treated chronic
lymphocytic leukemia patients with autoimmune cytopenias in the RESONATE
study. Blood Cancer J. 2017;7(2):e524. [PMC free article] [PubMed] [Google Scholar]
7. Milkins C, Berryman J, Cantwell C, et al.; British Committee for Standards in
Haematology. Guidelines for pre-transfusion compatibility procedures in blood
transfusion laboratories. Transfus Med. 2013;23(1):3-35. [PubMed] [Google Scholar]
8. Zö ller B, Li X, Sundquist J, Sundquist K. Risk of pulmonary embolism in patients
with autoimmune disorders: a nationwide follow-up study from Sweden. Lancet.
2012;379(9812):244-249. [PubMed] [Google Scholar]
9. Zö ller B, Li X, Sundquist J, Sundquist K. Risk of subsequent coronary heart disease
in patients hospitalized for immune-mediated diseases: a nationwide follow-up
study from Sweden. PLoS One. 2012;7(3):e33442. [PMC free
article] [PubMed] [Google Scholar]
10. Broome C, Cunningham JM, Mullins M, et al.. Incidence of thromboembolic events
is increased in a retrospective analysis of a large cold agglutinin disease (CAD)
cohort. Blood. 2017;130(suppl 1):928. [Google Scholar]
11. Hill A, Kelly RJ, Hillmen P. Thrombosis in paroxysmal nocturnal
hemoglobinuria. Blood. 2013;121(25):4985-4996, quiz 5105. [PubMed] [Google
Scholar]
12. Hill QA, Stamps R, Massey E, et al.. The diagnosis and management of primary
autoimmune haemolytic anaemia. Br J Haematol. 2017;176(3):395-411.
[PubMed] [Google Scholar]
13. Tjønnfjord E, Vengen OA, Berentsen S, Tjønnfjord GE. Prophylactic use of
eculizumab during surgery in chronic cold agglutinin disease. BMJ Case Rep.
2017;2017:bcr-2016-219066. [PMC free article] [PubMed] [Google Scholar]
14. Buckley L, Guyatt G, Fink HA, et al.. 2017 American College of Rheumatology
guideline for the prevention and treatment of glucocorticoid-induced
osteoporosis. Arthritis Care Res (Hoboken). 2017;69(8):1095-1110.
[PubMed] [Google Scholar]
15. Compston J, Cooper A, Cooper C, et al.; National Osteoporosis Guideline Group
(NOGG). UK clinical guideline for the prevention and treatment of
osteoporosis. Arch Osteoporos. 2017;12(1):43. [PMC free article] [PubMed] [Google
Scholar]
16. Kulpa J, Skrabs C, Simanek R, et al.. Probability of remaining in unsustained
complete remission after steroid therapy withdrawal in patients with primary
warm-antibody reactive autoimmune hemolytic anemia. Wien Klin Wochenschr.
2016;128(7-8):234-237. [PubMed] [Google Scholar]
17. Roumier M, Loustau V, Guillaud C, et al.. Characteristics and outcome of warm
autoimmune hemolytic anemia in adults: new insights based on a single-center
experience with 60 patients. Am J Hematol. 2014;89(9):E150-E155.
[PubMed] [Google Scholar]
18. Birgens H, Frederiksen H, Hasselbalch HC, et al.. A phase III randomized trial
comparing glucocorticoid monotherapy versus glucocorticoid and rituximab in
patients with autoimmune haemolytic anaemia. Br J Haematol. 2013;163(3):393-
399. [PubMed] [Google Scholar]
19. Michel M, Terriou L, Roudot-Thoraval F, et al.. A randomized and double-blind
controlled trial evaluating the safety and efficacy of rituximab for warm auto-
immune hemolytic anemia in adults (the RAIHA study). Am J Hematol.
2017;92(1):23-27. [PubMed] [Google Scholar]
20. Nakasone H, Kako S, Endo H, et al.. Diabetes mellitus is associated with high
early-mortality and poor prognosis in patients with autoimmune hemolytic
anemia. Hematology. 2009;14(6):361-365. [PubMed] [Google Scholar]
21. Lechner K, Jä ger U. How I treat autoimmune hemolytic anemias in adults. Blood.
2010;116(11):1831-1838. [PubMed] [Google Scholar]
22. Michel M, Godeau B, Aladjidi N, Perel Y Protocole national de diagnostic et de
soins (PNDS) Anémie Hémolytique Auto-Immune. Available at: https://www.has-
sante.fr/portail/jcms/c_2747976/fr/anemie-hemolytique-auto-immune-de-l-
enfant-et-de-l-adulte. Accessed 30 April 2018.
23. Reynaud Q, Durieu I, Dutertre M, et al.. Efficacy and safety of rituximab in auto-
immune hemolytic anemia: A meta-analysis of 21 studies. Autoimmun Rev.
2015;14(4):304-313. [PubMed] [Google Scholar]
24. Alonso HC, Manuel AV, Amir CG, et al.. Warm autoimmune hemolytic anemia:
experience from a single referral center in Mexico City. Blood Res. 2017;52(1):44-
49. [PMC free article] [PubMed] [Google Scholar]
25. Patel NY, Chilsen AM, Mathiason MA, Kallies KJ, Bottner WA. Outcomes and
complications after splenectomy for hematologic disorders. Am J Surg.
2012;204(6):1014-1019, discussion 1019-1020. [PubMed] [Google Scholar]
26. Salama A. Treatment options for primary autoimmune hemolytic anemia: a short
comprehensive review. Transfus Med Hemother. 2015;42(5):294-301. [PMC free
article] [PubMed] [Google Scholar]
27. Barcellini W, Fattizzo B, Zaninoni A, et al.. Clinical heterogeneity and predictors
of outcome in primary autoimmune hemolytic anemia: a GIMEMA study of 308
patients. Blood. 2014;124(19):2930-2936. [PubMed] [Google Scholar]
28. Thabet AF, Faisal M. Pulse cyclophosphamide therapy in refractory warm
autoimmune hemolytic anemia: a new perspective. Indian J Hematol Blood Transfus.
2014;30(4):313-318. [PMC free article] [PubMed] [Google Scholar]
29. Willis F, Marsh JC, Bevan DH, et al.. The effect of treatment with Campath-1H in
patients with autoimmune cytopenias. Br J Haematol. 2001;114(4):891-898.
[PubMed] [Google Scholar]
30. Passweg JR, Rabusin M, Musso M, et al.; Autoimmune Disease Working Party of
the EBMT. Haematopoetic stem cell transplantation for refractory autoimmune
cytopenia. Br J Haematol. 2004;125(6):749-755. [PubMed] [Google Scholar]
31. Barcellini W. New insights in the pathogenesis of autoimmune hemolytic
anemia. Transfus Med Hemother. 2015;42(5):287-293. [PMC free
article] [PubMed] [Google Scholar]
32. Wouters D, Stephan F, Strengers P, et al.. C1-esterase inhibitor concentrate
rescues erythrocytes from complement-mediated destruction in autoimmune
hemolytic anemia. Blood. 2013;121(7):1242-1244. [PubMed] [Google Scholar]
33. Desai J, Broome C. Complement blockade with C1 esterase inhibitor in severe
C3d positive autoimmune hemolytic anemia. Blood. 2016;128(22):4817. [Google
Scholar]
34. Marshall MJE, Stopforth RJ, Cragg MS. Therapeutic antibodies: what have we
learnt from targeting CD20 and where are we going? Front Immunol.
2017;8:1245. [PMC free article] [PubMed] [Google Scholar]
35. Miano M, Scalzone M, Perri K, et al.. Mycophenolate mofetil and sirolimus as
second or further line treatment in children with chronic refractory primitive or
secondary autoimmune cytopenias: a single centre experience. Br J Haematol.
2015;171(2):247-253. [PubMed] [Google Scholar]
36. Ratnasingam S, Walker PA, Tran H, et al.. Bortezomib-based antibody depletion
for refractory autoimmune hematological diseases. Blood Adv. 2016;1(1):31-
35. [PMC free article] [PubMed] [Google Scholar]
37. Mahévas M, Michel M, Vingert B, et al.. Emergence of long-lived autoreactive
plasma cells in the spleen of primary warm auto-immune hemolytic anemia patients
treated with rituximab. J Autoimmun. 2015;62:22-30. [PubMed] [Google Scholar]
38. Bussel J, Arnold DM, Grossbard E, et al.. Fostamatinib for the treatment of adult
persistent and chronic immune thrombocytopenia: Results of two phase 3,
randomized, placebo-controlled trials. Am J Hematol. 2018;93(7):921-930. [PMC
free article] [PubMed] [Google Scholar]
39. Kuter DJ, Arnold D, Lichtin A, et al. Fostamatinib, a spleen tyrosine kinase
inhibitor, for the treatment of warm antibody autoimmune hemolytic anemia:
preliminary results of the SOAR phase 2, multicenter, open-label study. Paper
presented at the 23rd Congress of the European Haematology Association; 25 June
2018; Copenhagen, Denmark. [Google Scholar]
40. Berentsen S, Ulvestad E, Gjertsen BT, et al.. Rituximab for primary chronic cold
agglutinin disease: a prospective study of 37 courses of therapy in 27
patients. Blood. 2004;103(8):2925-2928. [PubMed] [Google Scholar]
41. Rossi G, Gramegna D, Paoloni F, et al.. Short course of bortezomib in anemic
patients with relapsed cold agglutinin disease: a phase 2 prospective GIMEMA
study. Blood. 2018;132(5):547-550. [PubMed] [Google Scholar]
42. Rö th A, Bommer M, Hü ttmann A, et al.. Complement inhibition with eculizumab
in patients with cold agglutinin disease (CAD): results from a prospective phase II
trial (DECADE Trial). Blood. 2015;126(23):274. [Google Scholar]
43. Jaeger U, D’Sa S, Schoergenhofer C, et al.. Long term efficacy, safety and PK/PD
profile of the anti-C1s antibody (BIVV009) in primary cold agglutinin disease
patients. Blood. 2017;130(suppl 1):703. [Google Scholar]
44. Ricklin D, Barratt-Due A, Mollnes TE. Complement in clinical medicine: clinical
trials, case reports and therapy monitoring. Mol Immunol. 2017;89:10-21.
[PubMed] [Google Scholar]
45. Berentsen S, Randen U, Oksman M, et al.. Bendamustine plus rituximab for
chronic cold agglutinin disease: results of a Nordic prospective multicenter
trial. Blood. 2017;130(4):537-541. [PubMed] [Google Scholar]
46. Berentsen S, Randen U, Vå gan AM, et al.. High response rate and durable
remissions following fludarabine and rituximab combination therapy for chronic
cold agglutinin disease. Blood. 2010;116(17):3180-3184. [PubMed] [Google Scholar]